The basic rate-controlling mechanisms and fundamental stoichiometries of mitochondrial oxidative phosphorylation are very much in dispute. Previous work suggests that ADP-ATP translocation is rate-controlling, and this will be evaluated in mitochondria from various tissues utilizing an atractyloside titration technique. The hypothesis will be evaluated that the relative contributions of delta psi (membrane potential) and delta pH (pH gradient) to proton electrochemical gradient regulate ADP-ATP translocation during oxidative phosphorylation. Other experiments indicate that oxidative phosphorylation can approach overall thermodynamic equilibrium. If so, then ATP/2e- ratios cannot be equal for the three energy conserving sites. Moreover, ATP/O ratios should be higher for inverted membranes than for intact mitochondria, since the former do not require proton consuming ATP, ADP, and Pi translocation for ATP synthesis. These considerations necessitate a careful site by site re-evaluation of ATP/2e-, H ion/2e-, and H ion/ATP ratios. Utilizing firefly luciferase bioluminescence, optical probes, permeable ion and acid/base distributions, dual wavelength spectrophotometry, oxygen polarography, and coupled enzyme assays, near equilibrium measurements will be made of delta GR (free energy change of respiratory redox reactions, or redox potential), delta GP (free energy change of ATP synthesis, or phospate potential), and proton electrochemical gradient in order to estimate apparent ATP/O, H ion/O, H ion/ATP, H ion/site and ATP/site ratios for oxidative phosphorylation and pyridine nucleotide transhydrogenation by intact and inverted mitochondrial membranes. The results will be interpreted in terms of the theory of non-equilibrium thermodynamics and will be supplemented further by direct ATP/2e- measurements under level flow conditions. Other experiments will attempt to show complete reversibility of the reaction by demonstrating ATP driven reverse electron transport through cytochrome c oxidase. The proposed studies will provide essential new information concerning the basic kinetic control mechanisms and site by site stoichiometries of oxidative phosphorylation.